The insulin-releasing effects, cellular mechanisms of action and anti-hyperglycaemic activity of 10 analogues of esculentin-2CHa lacking the cyclic C-terminal domain (CKISKQC) were evaluated. Analogues of the truncated peptide, esculentin-2CHa(1–30), were designed for plasma enzyme resistance and increased biological activity. Effects of those analogues on insulin release, cell membrane integrity, membrane potential, intracellular Ca²⁺ and cAMP levels were determined using clonal BRIN-BD11 cells. Their acute effects on glucose tolerance were investigated using NIH Swiss mice. d-Amino acid substitutions at positions 7(Arg), 15(Lys) and 23(Lys) and fatty acid (l-octanoate) attachment to Lys at position 15 of esculentin-2CHa(1–30) conveyed resistance to plasma enzyme degradation whilst preserving insulin-releasing activity. Analogues, [d-Arg⁷,d-Lys¹⁵,d-Lys²³]-esculentin-2CHa(1–30) and Lys¹⁵-octanoate-esculentin-2CHa(1–30), exhibiting most promising profiles and with confirmed effects on both human insulin-secreting cells and primary mouse islets were selected for further analysis. Using chemical inhibition of adenylate cyclase, protein kinase C or phospholipase C pathways, involvement of PLC/PKC-mediated insulin secretion was confirmed similar to that of CCK-8. Diazoxide, verapamil and Ca²⁺ omission inhibited insulin secretion induced by the esculentin-2CHa(1–30) analogues suggesting an action on K_ATP and Ca²⁺ channels also. Consistent with this, the analogues depolarised the plasma membrane and increased intracellular Ca²⁺. Evaluation with fluorescent-labelled esculentin-2CHa(1–30) indicated membrane action, with internalisation; however, patch-clamp experiments suggested that depolarisation was not due to the direct inhibition of K_ATP channels. Acute administration of either analogue to NIH Swiss mice improved glucose tolerance and enhanced insulin release similar to that observed with GLP-1. These data suggest that multi-acting analogues of esculentin-2CHa(1–30) may prove useful for glycaemic control in obesity-diabetes.

The present study examines differences in metabolic and pancreatic islet adaptative responses following streptozotocin (STZ) and hydrocortisone (HC) administration in male and female transgenic Glu^CreERT2/Rosa26-eYFP mice. Mice received five daily doses of STZ (50 mg/kg, i.p.) or 10 daily doses of HC (70 mg/kg, i.p.), with parameters assessed on day 11. STZ-induced hyperglycaemia was evident in both sexes, alongside impaired glucose tolerance and reduced insulin concentrations. HC also had similar metabolic effects in male and female mice resulting in classical increases of circulating insulin indicative of insulin resistance. Control male mice had larger pancreatic islets than females and displayed a greater reduction of islet and beta-cell area in response to STZ insult. In addition, female STZ mice had lower levels of beta-cell apoptosis than male counterparts. Following HC administration, female mouse islets contained a greater proportion of alpha cells when compared to males. All HC mice presented with relatively comparable increases in beta- and alpha-cell turnover rates, with female mice being slightly more susceptible to HC-induced beta-cell apoptosis. Interestingly, healthy control female mice had inherently increased alpha-to-beta-cell transdifferentiation rates, which was decreased by HC treatment. The number of glucagon-positive alpha cells altering their lineage to insulin-positive beta cells was increased in male, but not female, STZ mice. Taken together, although there was no obvious sex-specific alteration of metabolic profile in STZ or HC mice, subtle differences in pancreatic islet morphology emphasises the impact of sex hormones on islets and importance of taking care when interpreting observations between males and females.

Discerning modification to the amino acid sequence of native glucagon can generate specific glucagon receptor (GCGR) antagonists, that include desHis¹Pro⁴Glu⁹-glucagon and the acylated form desHis¹Pro⁴Glu⁹(Lys¹²PAL)-glucagon. In the current study, we have evaluated the metabolic benefits of once-daily injection of these peptide-based GCGR antagonists for 18 days in insulin-resistant high-fat-fed (HFF) mice with streptozotocin (STZ)-induced insulin deficiency, namely HFF-STZ mice. Administration of desHis¹Pro⁴Glu⁹-glucagon moderately (P < 0.05) decreased STZ-induced elevations of food intake. Body weight was not different between groups of HFF-STZ mice and both treatment interventions delayed (P < 0.05) the onset of hyperglycaemia. The treatments reduced (P < 0.05–P < 0.001) circulating and pancreatic glucagon, whilst desHis¹Pro⁴Glu⁹(Lys¹²PAL)-glucagon also substantially increased (P < 0.001) pancreatic insulin stores. Oral glucose tolerance was appreciably improved (P < 0.05) by both antagonists, despite the lack of augmentation of glucose-stimulated insulin release. Interestingly, positive effects on i.p. glucose tolerance were less obvious suggesting important beneficial effects on gut function. Metabolic benefits were accompanied by decreased (P < 0.05–P < 0.01) locomotor activity and increases (P < 0.001) in energy expenditure and respiratory exchange ratio in both treatment groups. In addition, desHis¹Pro⁴Glu⁹-glucagon increased (P < 0.01–P < 0.001) O₂ consumption and CO₂ production. Together, these data provide further evidence that peptidic GCGR antagonists are effective treatment options for obesity-driven forms of diabetes, even when accompanied by insulin deficiency.

Xenin-25, a peptide co-secreted with the incretin hormone glucose-dependent insulinotropic polypeptide (GIP), possesses promising therapeutic actions for obesity-diabetes. However, native xenin-25 is rapidly degraded by serum enzymes to yield the truncated metabolites: xenin 9–25, xenin 11–25, xenin 14–25 and xenin 18–25. This study has examined the biological activities of these fragment peptides. In vitro studies using BRIN-BD11 cells demonstrated that native xenin-25 and xenin 18–25 possessed significant (P<0.05 to P<0.001) insulin-releasing actions at 5.6 and 16.7 mM glucose, respectively, but not at 1.1 mM glucose. In addition, xenin 18–25 significantly (P<0.05) potentiated the insulin-releasing action of the stable GIP mimetic (d-Ala²)GIP. In contrast, xenin 9–25, xenin 11–25 and xenin 14–25 displayed neither insulinotropic nor GIP-potentiating actions. Moreover, xenin 9–25, xenin 11–25 and xenin 14–25 significantly (P<0.05 to P<0.001) inhibited xenin-25 (10⁻⁶ M)-induced insulin release in vitro. I.p. administration of xenin-based peptides in combination with glucose to high fat-fed mice did not significantly affect the glycaemic excursion or glucose-induced insulin release compared with controls. However, when combined with (d-Ala²)GIP, all xenin peptides significantly (P<0.01 to P<0.001) reduced the overall glycaemic excursion, albeit to a similar extent as (d-Ala²)GIP alone. Xenin-25 and xenin 18–25 also imparted a potential synergistic effect on (d-Ala²)GIP-induced insulin release in high fat-fed mice. All xenin-based peptides lacked significant satiety effects in normal mice. These data demonstrate that the C-terminally derived fragment peptide of xenin-25, xenin 18–25, exhibits significant biological actions that could have therapeutic utility for obesity-diabetes.

Recently, glucagon-like peptide 1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP) have received much attention regarding possible roles in aetiology and treatment of type 2 diabetes. However, peptides co-secreted from the same enteroendocrine cells are less well studied. The present investigation was designed to characterise the in vitro and in vivo effects of xenin, a peptide co-secreted with GIP from intestinal K-cells. We examined the enzymatic stability, insulin-releasing activity and associated cAMP production capability of xenin in vitro. In addition, the effects of xenin on satiety, glucose homoeostasis and insulin secretion were examined in vivo. Xenin was time dependently degraded (t _1/2=162±6 min) in plasma in vitro. In clonal BRIN-BD11 cells, xenin stimulated insulin secretion at 5.6 mM (P<0.05) and 16.7 mM (P<0.05 to P<0.001) glucose levels compared to respective controls. Xenin also exerted an additive effect on GIP, GLP1 and neurotensin-mediated insulin secretion. In clonal β-cells, xenin did not stimulate cellular cAMP production, alter membrane potential or elevate intra-cellular Ca^{2 +}. In normal mice, xenin exhibited a short-acting (P<0.01) satiety effect at high dosage (500 nmol/kg). In overnight fasted mice, acute injection of xenin enhanced glucose-lowering and elevated insulin secretion when injected concomitantly or 30 min before glucose. These effects were not observed when xenin was administered 60 min before the glucose challenge, reflecting the short half-life of the native peptide in vivo. Overall, these data demonstrate that xenin may have significant metabolic effects on glucose control, which merit further study.

In this work, our aim was to determine whether l-arginine (a known insulinotropic amino acid) can promote a shift of β-cell intermediary metabolism favoring glutathione (GSH) and glutathione disulfide (GSSG) antioxidant responses, stimulus–secretion coupling and functional integrity. Clonal BRIN-BD11 β-cells and mouse islets were cultured for 24 h at various l-arginine concentrations (0–1.15 mmol/l) in the absence or presence of a proinflammatory cytokine cocktail (interleukin 1β, tumour necrosis factor α and interferon γ). Cells were assessed for viability, insulin secretion, GSH, GSSG, glutamate, nitric oxide (NO), superoxide, urea, lactate and for the consumption of glucose and glutamine. Protein levels of NO synthase-2, AMP-activated protein kinase (AMPK) and the heat shock protein 72 (HSP72) were also evaluated. We found that l-arginine at 1.15 mmol/l attenuated the loss of β-cell viability observed in the presence of proinflammatory cytokines. l-Arginine increased total cellular GSH and glutamate levels but reduced the GSSG/GSH ratio and glutamate release. The amino acid stimulated glucose consumption in the presence of cytokines while also stimulating AMPK phosphorylation and HSP72 expression. Proinflammatory cytokines reduced, by at least 50%, chronic (24 h) insulin secretion, an effect partially attenuated by l-arginine. Acute insulin secretion was robustly stimulated by l-arginine but this effect was abolished in the presence of cytokines. We conclude that l-arginine can stimulate β-cell insulin secretion, antioxidant and protective responses, enabling increased functional integrity of β-cells and islets in the presence of proinflammatory cytokines. Glucose consumption and intermediary metabolism were increased by l-arginine. These results highlight the importance of l-arginine availability for β-cells during inflammatory challenge.

Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human β-cell line. Structural pseudoislets formed readily over 3–7 days in culture using ultra-low-attachment plastic, attaining a static size of 100–200 μm in diameter, corresponding to ∼6000 β cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1.7- to 12.5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human β cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.

Interleukin-6 (IL6) has recently been reported to promote insulin secretion in a glucagon-like peptide-1-dependent manner. Herein, the direct effects of IL6 (at various concentrations from 0 to 1000 pg/ml) on pancreatic β-cell metabolism, AMP-activated protein kinase (AMPK) signaling, insulin secretion, nitrite release, and redox status in a rat clonal β-cell line and mouse islets are reported. Chronic insulin secretion (in μg/mg protein per 24 h) was increased from 128.7±7.3 (no IL6) to 178.4±7.7 (at 100 pg/ml IL6) in clonal β-cells and increased significantly in islets incubated in the presence of 5.5 mM glucose for 2 h, from 0.148 to 0.167±0.003 ng/islet. Pretreatment with IL6 also induced a twofold increase in basal and nutrient-stimulated insulin secretion in subsequent 20 min static incubations. IL6 enhanced both glutathione (GSH) and glutathione disulphide (GSSG) by nearly 20% without changing intracellular redox status (GSSG/GSH). IL6 dramatically increased iNOS expression (by ca. 100-fold) with an accompanying tenfold rise in nitrite release in clonal β-cells. Phosphorylated AMPK levels were elevated approximately twofold in clonal β-cells and mouse islet cells. Calmodulin-dependent protein kinase kinase levels (CaMKK), an upstream kinase activator of AMPK, were also increased by 50% after IL6 exposure (in β-cells and islets). Our data have demonstrated that IL6 can stimulate β-cell-dependent insulin secretion via direct cell-based mechanisms. AMPK, CaMKK (an upstream kinase activator of AMPK), and the synthesis of nitric oxide appear to alter cell metabolism to benefit insulin secretion. In summary, IL6 exerts positive effects on β-cell signaling, metabolism, antioxidant status, and insulin secretion.

Asparagus racemosus root has previously been reported to reduce blood glucose in rats and rabbits. In the present study, the effects of the ethanol extract and five partition fractions of the root of A. racemosus were evaluated on insulin secretion together with exploration of their mechanisms of action. The ethanol extract and each of the hexane, chloroform and ethyl acetate partition fractions concentration-dependently stimulated insulin secretion in isolated perfused rat pancreas, isolated rat islet cells and clonal β-cells. The stimulatory effects of the ethanol extract, hexane, chloroform and ethyl acetate partition fractions were potentiated by glucose, 3-isobutyl-1-methyl xanthine IBMX, tolbutamide and depolarizing concentration of KCl. Inhibition of A. racemosus-induced insulin release was observed with diazoxide and verapamil. Ethanol extract and five fractions increased intracellular Ca²⁺, consistent with the observed abolition of insulin secretory effects under Ca²⁺-free conditions. These findings reveal that constituents of A. racemosus root extracts have wide-ranging stimulatoryeffects on physiological insulinotropic pathways. Future work assessing the use of this plant as a source of active components may provide new opportunities for diabetes therapy.